Genetic engineering is a powerful approach to the manipulation of proteins. However, genetic methodologies are constrained by the use of only naturally coded amino acids. Furthermore, cytotoxic proteins are difficult to obtain by expression and isolation from a living source, since the expression of the toxic protein can result in death of the host.
To some extent, protocols have been developed to circumvent these problems, for example, total chemical synthesis (Kent, S. B. (1988) Ann. Rev. Biochem. 57:957-989), use of misacylated tRNAs (Noren, et al., (1989) Science 244:182-188), and semi-synthetic techniques (reviewed in Offord, R. (1987) Protein Eng. 1:151-157; Roy. et al. (1994) Methods in Enzymol. 231:194-215; Wallace, C. J. (1993) FASEB 7:505-515). However, all of these procedures are limited by either the size of the fragment which can be generated or by low reaction yield.
It would therefore be desirable to develop a high-yield, semi-synthetic technique to allow in vitro fusion of a synthetic protein or peptide fragment to an expressed protein without limitation as to the size of the fused fragments.
Likewise, in order to produce cytotoxic proteins, it would be desirable to develop a method of fusing a synthetic fragment, in vitro, to an inactive, expressed protein, so as to restore protein activity post-production from the host.
The modified Sce VMA intein has been used to generate thioester-tagged proteins for use in ligation (Example 19, U.S. Ser. No. 08/811,492, filed Jun. 16, 1997; Chong, (1996) J. Biol. Chem., 271(36):22159-22168; Chong, (1997) Gene, 192:271-281; and Muir, et al. (1998) Proc. Natl. Acad. Sci USA 95:6705-6710).
Some disadvantages have been low yields due to poor cleavage of the Sce VMA intein with thiol-reagents that are optimum for ligation, the need for large peptide quantities due to on-column reactions, the use of odoriferous reagents, and/or low protein yields due to the use of a large, eukaryotic intein.